This invention relates to a method for distinguishing among bacteria within the same taxonomic group based on the reactivity of specific 16S subsequences found within the ribosomal operons of the organisms, using probes during hybridization under conditions of increasing severity (stringency). Hybridization is the process whereby two strands of nucleic acid can interact and, if sufficiently matched in sequence, form a double-stranded structure. By the term probe is meant a marked, single-stranded nucleic acid sequence that is complementary to the nucleic acid sequences to be detected (target sequences). The use of this method of operon analysis for distinguishing the genera Escherichia from Shigella and for distinguishing among species of Shigella is demonstrated together with nucleic acid probes needed for conducting the analyses.
The terms xe2x80x9cEscherichia coli,xe2x80x9d xe2x80x9cShigella boydii,xe2x80x9d xe2x80x9cShigella dysenteriae,xe2x80x9d xe2x80x9cShigella flexneri,xe2x80x9d and xe2x80x9cShigella sonneixe2x80x9d refer to the bacteria classified as such in Bergey""s Manual of Determinative Bacteriology, 8th Edition, R. D. Buchanan and N. E. Gibbons, Eds., William and Wilkins, 1974, pp. 290-339. Unless specified otherwise the term Shigella will refer collectively to the four species mentioned above.
Detection of Shigella is important for medical diagnosis, public health surveillance, food safety, and other applications. Cases of Shigella, which must be identified by species, are required to be reported to the Centers for Disease Control and Prevention, which tracks the incidence and prevalence of Shigella in every state in the United States and in the District of Columbia. Current methods of detection are neither simple, straightforward, nor absolute (J. J. Farmer, III and M. T. Kelly, Enterobacteriaceae, in A. Balows, Ed, Fifth Edition, Manual of Clinical Microbiology, Washington, D.C., American Society for Microbiology, 1991.)
Suspected colonies usually are grown on both MacConkey agar and xylose-lysine-deoxycholate agar. Colonies of Shigella missed on one medium may show up on the other. Many laboratories also use Hektoen enteric agar. Enrichment of less than optimum cultures may require GN broth. Selenite broth may be useful for isolating S. sonnei. Suspected colonies of Shigella require confirmation by culture on other types of growth media such as triple sugar iron or Kliger iron agar slants. Colonies that show an alkaline/acid reaction with no H2S or gas then must be screened further by serological analysis with antisera in order to identify each of the species of Shigella.
Even with these procedures, differentiating strains of Shigella from Escherichia coli has proved to be one of the most difficult problems for a clinical microbiology laboratory. Recommended guidelines are complicated by exceptions related to one or more Shigella species. The difficulties inherent in distinguishing these organisms often forces investigators to depend merely on the fact that two Shigella species (S. boydii and S. flexneri) are not as prevalent in the United States, although a significant number of cases do occur, as are the other two species in order to help solidify their diagnoses. Still, the guidelines conclude with the realization that no definitive rules on the identification of Shigella isolates can be made and complete biochemical and serological typing must be done in each instance.
It is yet another aspect of the invention to avoid the disadvantages associated with the traditional culturing and serological identification techniques and to employ nucleic acid probes to distinguish Escherichia coli from Shigella and to identify each of its four associated species.
Efforts to circumvent the difficult, expensive, and time-consuming procedures with a simple yet rapid molecular procedure for differentiating the genus Shigella from the genus Escherichia and for separately identifying each of the four individual species of Shigella has also proved difficult. This has been attributed, in particular, to the very close relatedness of E. coli and all four Shigella species by DNA-DNA hybridization (J. J. Farmer, III and M. T. Kelly, Enterobacteriaceae, in A. Balows, Ed, Fifth Edition, Manual of Clinical Microbiology, Washington, D.C., American Society for Microbiology, 1991.)
Kyriaki Parados and Janice McCarty (U.S. Pat. No. 5,648,481) have identified a set of nucleic acid probes for detection of the genus Shigella and/or E. coli (EIBC) based on specific chromosomal sequences and fragments of Shigella. These probes can neither distinguish between E. coli and Shigella nor can they separately distinguish between one species of Shigella and another. In addition, the relatively large probes (approximately 40 nucleotides each) require hybridization overnight followed by exposure for 15 hours to x-ray film to produce autoradiographs.
Alessio Fasano, Myron M. Levine, James P. Nataro, and Fernando Noriega (U.S. Pat. No. 5,589,380) targeted the enterotoxins of Shigella flexneri2a and produced antibodies to the same, which might be useful primarily for the identification of S. flexneri. Kyriaki Parodos, Hsien-Yeh Hsu, Daid Sobell, Janice M. McCarty, and David J. Lane (U.S. Pat. No. 5,084,565) devised probes capable of hybridizing to rRNA of both E. coli and Shigella species but unable to discriminate among them. Phillippe Sansonetti, Catherine Boileau and Hxc3xa9lxc3xa8ne D""Hauteville (U.S. Pat. No. 4,992,364) targeted the 140 MDalton virulence plasmid of S. flexneri. Their probes are relatively large, ranging in size from about 11.5 kbases to 27 kbases, and identify only combined strains of Shigella and E. coli carrying the virulence plasmid. Long-term hybridization (overnight) is followed by 6 hours of exposure to produce autoradiographs.
A subsequence of ribosomal RNA (rRNA) or its gene presents a potential target for separate identification of E. coli and each of the four species of Shigella through hybridization with appropriate DNA or RNA probes. Portions of rRNA have been found not to be conserved among diverse bacterial species, making them potential hybridization targets for distinguishing between one taxonomic group and another. David E. Kohne (U.S. Pat. No. 5,601,984) discusses such a method for detecting and quantitating organisms. But Kohne does not provide the teaching necessary to make Shigella species-specific probes.
Furthermore, Kohne does not teach how to distinguish among very closely related organisms using probes where a subsequence of a rRNA subunit or rRNA subunit gene is not specific to the taxonomic group (qualitative difference) but rather occurs as multiple but slightly differentiated copies in different proportions among multiple operons for the RNA genes (quantitative difference). An operon is defined as a group of contiguous genes that are coordinately regulated by controlling elements. Nor does Kohne teach the use of probes of sequence specific neither to genus nor species or other taxonomic grouping.
The E. coli chromosome is circular and contains seven operons for rRNA (FIG. 1). A typical rRNA (rrn) operon contains two promoters and genes for 16S, 23S, and 5S rRNA and a single 4S tRNA gene (FIG. 2). When analyzed, the 16S genes of the different E. coli rrn operons have been found to have regions where the sequences have been altered through mutations (Table 1). In some operons the mutations are the same in one particular region and in other operons they are different. Other organisms such as Shigella may either have a different number of operons, different types of operons, a different proportion of a particular mutation in one or more of its operons, one or more mutations in its operons distinct from E. coli or all of these possibilities.
FIG. 1 is an illustration of the ribosomal RNA operon on the E. coli chromosome. Each line marks the relative positions of one of the seven rrn operons found on the E. coli chromosome.
FIG. 2 is an illustration of a ribosomal RNA operon.
The number of possible potential combinations suggests that hybridization of rrn operon subunit probes specific for 16S does not detect subsequences specific to a taxonomic unit but instead detects a variable number of a targeted mutation within the operons, the number and variability of which may differ from one closely related organism to another. Thus, the rrn operon subsequences for 16S not only exhibit phylogenetic variability between organisms but variability even within a given genus and species of a single organism.
Sequencing of 16S genes in rrn operons does not necessarily clarify the variability seen. The entire process of preparing a plasmid vector containing ligated genetic material from rrn operon 16S genes in order to conduct the sequencing studies fails generally to select either for a specific operon or for a single bacterial cell containing a vector carrying a single rrn operon. DNA representing the rrn operon 16S subsequences, for example, prepared from Shigella is amplified in the polymerase chain reaction (PCR) using universal primers that are unlikely to discriminate between subsequences from one operon and another. Competent cells carrying the ligated vector are then selected for large-scale cloning. A colony of cells, rather than a single cell or cell clone, is chosen.
In all cases of selection for the sequencing process, heterogenous rrn operon material rather than homogenous rrn operon nucleic acid may be obtained. The resulting genetic sequences may either then represent a broad consensus sequence from all the possible rrn operon subsequences or a skewed sequence representing some but not all of the rrn operons and their different subsequences. This makes it very difficult to predict from the rrn sequences which probe subsequences are most likely to differentiate between one taxonomic group and another.
Hybridization refers to the process whereby sequence-specific, base-paired duplexes from any combination of nucleic acid fragments are formed. A useful measure of the stability of a DNA duplex or an RNA-DNA hybrid is the melting temperature or Tm, which refers to the temperature at which the strands are half dissociated or denatured. Complexity refers to the total length of different sequences present in a sample of nucleic acid. Reassociation is the process of joining together by typical base pairing the two fully separated complementary sequences. Complementary refers to rules of base pairing enunciated by Erwin Chargaff whereby an adenine base pairs with its thymine complement and a guanine base pairs with its cytosine complement.
Hybridization reactions are usually done in a buffered, aqueous medium that may contain other additives. Additives may include detergent, salts, polymers, and blocking agents. The stringency of the hybridization medium may be controlled by temperature, salt concentration, probe concentration, probe length, time, and other factors. The rate of reassociation of two simple DNA strands with complementary sequences and no significant sequence repetition is easily described by practical kinetic equations such as (1) and (2) given below.
H=(1+kCot)xe2x88x921xe2x80x83xe2x80x83(1)
where H=fraction of DNA not bound to hydroxyapatite, k=observed rate constant, Co=original concentration of nucleotides, and t=time in seconds. Hydroxyapatite is used to distinguish single-stranded DNA from double-stranded DNA.
S=(1+kCot)xe2x88x920.44xe2x80x83xe2x80x83(2)
where S=fraction of nucleotides remaining unpaired based on the use of nuclease S1 to differentiate single and double-stranded DNA.
The kinetics describing the rate of reassociation for complex genomes with sequence repetition is more difficult, particularly if bound to a filter or other surface such as an optic fiber. Although bacteria have relatively simple genomes compared to higher organisms, the repetition and multiplicity of the rrn operons in bacteria make their hybridization reactivity difficult to predict. For example, as stringency is increased, sequences that are not perfectly complementary should become less stable. The extent of hybridization of a probe to a given bacterial genome, therefore, might be expected to decrease with increasing temperature if there were some mismatching of bases or, at best, stay the same if there was perfect complementarity.
Stringency washes are usually performed at 3-5xc2x0 C. below the Tm of the perfectly matched probe when differentiation from mismatched sequences is required. The Wallace rule (R. B. Wallace and C. G. Miyada, Methods in Enzymology 152:438, 1990) can be used as follows to calculate the Tm for a probe in order to set the temperature conditions necessary to avoid mismatched sequences:
Tm=(4xc3x97 number of G+C bases)+(2xc3x97 number of A+T bases)xe2x80x83xe2x80x83(3)
However, when the Tm is calculated in this matter for each of the probes of the present invention, it is found to be more than 5xc2x0 below the actual Tm as measured in 1 M NaCl. This discrepancy further complicates a determination of stringency for rrn operon analysis. The method of the present invention provides conditions for hybridization of the probes that exceeds the calculated Tm and ensures that stringent conditions are being employed.
Previous studies have shown that a bell-shaped curve describes the relationship between the rate of hybridization and the temperature of incubation for formation of well-matched hybrids (Margaret L. M. Anderson and Bryan D. Young, Quantitative Filter Hybridization, in Roy J. Britten and Eric H. Davidson, Nucleic Acid Hybridization, Academic Press, 1985). The curves show a low relative rate of reassociation of perfectly matched sequences at either end (xe2x88x9250xc2x0 C. below the Tm and at the Tm) of the bell curve and a maximal rate of reassociation at xe2x88x9220xc2x0 C. below the Tm. The rate of reassociation for mismatched sequences falls to zero at xe2x88x9220xc2x0 C. below the Tm.
Such studies do not teach the hybridization reactivity seen when the probes of the present invention directed toward rrn operon 16S subsequences are hybridized at temperatures above their calculated Tm. The studies do not generally predict a loss of hybridization reactivity at temperature a few degrees below the measured Tm followed by a reappearance of hybridization reactivity as the temperature approaches the measured Tm of the probe. This phenomenon is seen, however, for some but not all of the probes used to test E. coli and the Shigella species reported herein. The reactivity of these particular probes with bacterial genomes have neither been previously disclosed or discussed.
The present invention also provides a method for organizing the complexity of probe reactivity with E. coli and Shigella organisms into a hierarchical flow diagram for identifying one or more of these closely related organisms when present in a sample.
In another aspect, the present invention contemplates a diagnostic kit for screening a test sample for the presence of Shigella species or E. coli. Such a kit would contain a nucleic acid probe having specificity for a species specific or genus specific nucleotide.